The present invention is related to the field of medicine, particularly to the development of new formulations for immunological potentiation allowing the increase of the amount and quality of the immune response to vaccine antigens.
The technical aim of the invention is the development of formulations that are capable of increasing the levels of the immune response of the body to vaccine antigens.
The adjuvants are substances that increase or optimize the immune response to antigens inoculated through the mucosal or systemic routes. The adjuvants or their formulations are combined with the antigen to generate or potentiate the type of response desired, decrease the number of inoculations and reduce the amount of antigen needed to obtain and maintain protection (McElrath, M. C. 1995 Seminars in Cancer Biology 6: 375-385).
Adjuvants have been developed as a necessity due to the advancements of modern biotechnology with the production of pure soluble recombinant and synthetic antigens. In general, these antigens are safe, but generally shows a decreased immunogenicity compared to those of the original organism. An important function of adjuvants or their formulations, is to increase the complexity of the antigen facing the immune system, in a safe way, thus increasing its immunogenicity (Alving, R. C. 1992 AIDS Res. Hum. Retroviruses 8 (8): 1427-1430).
At present, the search for new adjuvants and immunological stimulators, as well as the development of new ways of delivering antigens and pharmaceuticals, is one of the lines of world research in the pharmaceutical field, especially in vaccines. The development of the adjuvants for mucosal use is a present need in the vaccine field (Report of the Expert Panel VI: Concerted efforts in the field of mucosal immunology 1996 Vaccine 14: 644-664). They may be classified as mucosal and systemic adjuvants, considering that the physiological characteristics on receiving and processing the antigen in both inoculation routes generate different adjuvation procedures. The mucosal route, according to the characteristics of the antigen, require binding or coating procedures with specific ligands that send the antigens to the M cells. The adjuvant activity for mucosal antigens is obtained through strategies that help the antigen to cross the borders imposed by the route. The physical characteristics of the antigen may favor its phagocytosis. Once the antigen has been assimilated, the adjuvant may influence the response by any of the known mechanisms: antigen adsorption, depot effect, cytokine induction, complement activation, recruiting of different cell populations of the immunological system, antigen delivery to different antigen presenting cells, regulation of the expression through class I or class II and the stimulation to produce different antibody subtypes (McElrath M. C. 1995 Seminars in Cancer Biology 6: 375-385).
Some of the immunological stimulators known, as muramyldipeptides (MDP), monophosphoryl-lipid A (MPL), the lipoid amine Avridine and those known as toxins: V. cholerae (CT) and of E. coli (HLT) toxins, are recognized adjuvants for antigens administered through the mucous route (Walker, R. I. 1994 Vaccine 12 (5): 387-400).
The MDP and MPL have been studied in liposomal formulations for therapeutic and prophylactic use, however, the toxins and their sub-units (specially the CT and CTB) are the most common mucosal adjuvants.
The ability of CT to act as an oral adjuvant has been confirmed by a large number of researchers (McGhee, J. R. et al. 1992 Vaccine 10 (2): 75-88). The Cholera Toxin does not fulfill the classical definition of an adjuvant because it stimulates an immune response against itself and its adjuvant activity depends on its immunogenicity (Elson, C. O. 1987 Fed. Proc. 46: 1778). The immunomodulating effects of the CT and HLT explaining their strong adjuvant activity, include the increase in antigen presentation by several types of B cells, the increase in B cells differentiation to the IgA isotype, the interaction with T cells and the induction of cytokine production (Lintermans, P. 1995 Advanced Drug Delivery Reviews 18: 73-89).
From the practical point of view, the use of the holotoxin in man is not possible due to its toxicity. A better strategy is the detoxification of the CT, by separating the CTA subunit or through mutations of the gene codifying it. CT as well as the CTB (less toxic subunit of CT) may potentiate the immune response to several antigens bound covalently through specific interactions with M cells (Holmgren, J. et al. 1993 Vaccine 11: 1179-1184).
The antigen delivery systems have reached a sufficiently high degree of development as to have an impact on immunization. It is expected that the solid particulated systems for parenteral or non-parenteral administration should be among the first licensed products (Li Wan Po et al. 1995 Advanced Drug Delivery Reviews 18: 101-109).
Through the possibilities offered by the antigen delivery systems in the particulation of soluble antigens, and taking advantage of the physiological characteristics of the mucosal route, these systems have been tested and have shown adjuvant activity. In the literature they have been classified as:
a) synthetic/inactivated and b) alive (Report of the Expert Panel VII: Vaccine Delivery Systems 1996 Vaccine 14: 644-664). Enclosed in the first group, according to their non-living characteristic, there are two different groups. In respect to the first group, the artificial polymeric particles have been studied with different results comprising: the co-polymeric microspheres of lactic and glycolic acids, also alternative polymers as polyphosphacenes, cellulose acetate polymers, iminocarbonates, ethylenvinyl acetate polymers, proteinoid microspheres, dextran polyanhydrid and nanospheres; the particles produced from natural materials: alginates, gelatins and seeds of plants and also the liposomes and their variants: proteoliposomes, virosomes and ISCOMs (Li Wan Po et al. 1995 Advanced Drug Delivery Reviews 18: 101-109).
The size of the particles is within the group of important factors for antigen sending. In the case of the mucosal immunization route it has been reported that particles of a diameter greater than 10 xcexcm are not absorbed (Eldridge J. H. 1990 J. Control. Release 11:205-214). In experiments with rats it was observed that after oral administration, only particles of 5 xcexcm deeply penetrated the Peyer Patches and those of 1 xcexcm of diameter, penetrated the lymphonodes and went into the bloodstream (Jani, P. et al 1990 J. Pharm. Pharmacol. 42:821-826)(Alpar, H. O. et al. 1989 J. Pharm. Pharmacol 41:194-196).
The extrapolation of these results in man is not yet defined and sometimes the adsorption by the gastrointestinal tract is not a requirement for adjuvant activity although it has been proven that with the adsorption of the diphtheria toxoid in plant seed of up to 2 mm diameter, the immune response was potentiated (Mirchamski, H. et al. 1994 Vaccine 12:1167-1172).
The determination of the optimum size of the controlled release systems through the oral, rectal or vaginal routes is under study (Li Wan Po et al. 1995 Advanced Drug Delivery Reviews 18:101-109).
Another factor affecting the particles is the hydrophobic-hydrophilic balance, which may be modified to obtain a modulation of the immune response (Jani, P. et al 1990 J. Pharm. Pharmacol. 42:821-826).
Recently, the use of vaccines of protein cochleates described in 1975 (Papahadjopoulos D. et al 1975 Biochem. Biophys. Acta 394: 483), has been patented. The cochleates are complexes of liposomes and divalent cations (mainly calcium) that, through the calcium-phospholipid interaction, enable the formation of a liposome structure coiled on itself, allowing the immunization through different routes (Gould Foguerite, S. WO 95/09648). With this structure, the immune response of antibodies, as well as the cell mediated responses are stimulated (Gould Foguerite, S. 1994 AIDS Res. Hum. Retroviruses 10 (Supl. 2): S99-S103).
The absorption and passage of the antigens through specialized cells of the epithelium associated to the lymphoid follicle, the M cells is a critical step in the generation of an immune response through the simple intestinal epithelium, which is generally accepted by all authors. Through this process, the antigens are sent to the basal pocket of the M cells, constituting the first contact of the antigen (practically undegraded) with B and T cells and macrophages. The main function of the pocket is to provide the organism with an environment protected from the modulator influence of the external humoral factors (Neutra, R. M. 1996 Annu. Rev. Immunol. 14: 275-300).
In the respiratory tract, the epithelium varies from pseudostratified, to simple. In the bronchi, the simple epithelial zone, the intercellular spaces are sealed by tight binds and the main mechanism for the antigens to enter is through the M cells. At the tonsils, the predominant epithelium is the stratified epithelium. Here, the absorption mechanism of the antigen is closely associated to a net of macrophages and mobile dendritical cells from the bone marrow, of up to 700 cells per mm2. These cells are able to migrate to the organized lymphoid tissue associated to the mucous (O-MALT) or to a lymphonode, presenting the processed antigen that was phagocyted at the surface of the tonsils. Under normal conditions this constitutes the main mechanism for the presentation of the antigens through MHC class II of the respiratory tract (Neutra, R. M. 1996 Annu. Rev. Immunol. 14: 275-300).
The difference in the absorption of the antigen at the oral and nasopharyngeal levels allows to understand why the inoculation of an adjuvant through both routes will not necessarily produce the same results. Therefore, it is not obvious that an adjuvant that is effective through the oral route, will or will not be effective by the nasopharyngeal route. The adjuvants may have different effects in different mucous sites, since different mucous surfaces have different micro-environments. The progress in the knowledge of the physiology of the mucous and the mode of adjuvants action may help to the development of more effective mucosal vaccines. As well as the characteristics of the antigen as: size, presence of mucosal ligands, electric charge, lipophilicity and T dependence may also affect the immune response. (Report of the Expert Panel VI: Concerted efforts in the field of mucosal immunology 1996 Vaccine 14: 644-664). At the same time, other elements of the organism as the hormonal fluctuations during the menstrual cycle, affect the assimilation of the antigen at the vagina. This fact shows the strong collaboration between the dendritic and epithelial cells and explains the variation in the effectiveness of the vaccines through the vaginal route (Parr, E. L.,Parr, M. B. 1992 Vaccine Res. 1: 221-25).
For the case of the simple epithelial route of the intestine, the delivery of non-living vaccines to the M cells has been difficult, given the fact that the unprotected macromolecules are readily digested or dragged by the secretions and the motility of the gastrointestinal tract. There is little information available in relation to the components of the apical membrane of the M cells that may serve as receptors. The liposomes and the micro-particles may adhere to the mucous surfaces by hydrophobic interactions, but the entrance of the antigens to the M cells is inefficient since they are rapidly trapped in the mucous gels and many do not reach the mucous. The macromolecules or particles conjugated or coated with ligands as the CTB, have the limiting factor of their access to the receptors (Neutra, R. M. 1996 Cell 86: 345-348).
Recent experiments in mice, using 28.8 nm coloidal gold particles coated with CTB showed that they were able to adhere and penetrate selectively in M cells of the epithelium associated to the mucous follicle, being unable to penetrate the enterocytes of the so-called xe2x80x9cbrush borderxe2x80x9d. Larger particles; of approximately 1.13 xcexcm; also coated with CTB, were unable to adhere to M cells, while CTB-FITC particles of approximately 6.4 nm, adhered and entered through both cell types. This experiment demonstrated the role of the glycocalix in the adherence and entering of the antigens through the M cells. The B subunit of the cholera toxin has receptors both in the enterocytes as in the M cells. The use of the ligands bound to particles may result in a specific adherence to M cells, but only in a range of sizes restricted by the glycocalix. Particles of 1 xcexcm or greater, require ligands that are specifically directed to components of the M cells. The identification of these components is still under study (Frei, A. et.al. 1996 J. Exp. Med. 184: 1045-1059).
A group of pathogenic bacteria is able to bypass the difficulties of the non-living systems on being efficiently assimilated by the lack of receptors at the M cells. These bacteria exploit this mechanism to infect mucosal tissues and disseminate themselves systemically before being detained by the immune system. The bacterial pathogens that adhere to the surface of the M cells start the signal transduction events at the epithelial level that promote their entrance. The best known one is the attenuated bacterial strains of S. typhi ty21a, for which a lectin type interaction with a receptor of polysaccharidic nature exist on the cell membrane of M cells. Also safe and effective are the attenuated live strains of V. cholerae and polio virus for oral immunization. The genetically manipulated strains of these microorganisms were first used in man as carriers of heterologous antigens (Mekalanos, J. J. 1992 Adv. Exper. Med. Biol. New York Plenum Press: 43-50).
The biology of these live vectors introduce new challenges. The vaccine strains of V. cholerae, that have no genes for the toxin, may still produce diarrhea, seemingly because the epithelial cells release cytokines as a response to bacterial adherence (Mekalanos, J. J. 1992 Adv. Exper. Med. Biol. New York Plenum Press: 43-50). The main challenge in the use of the genetically manipulated strains of the S. typhi and S. typhimurium consist in obtaining a sufficient attenuation offering safety while still adhering to the M cells and their proliferation in mucosal tissues, to maintain their immunogenicity. The attenuated strains of Shigella, are also internalized by the M cells, but they have lost their ability to disseminate from cell to cell. This phenomenon is the basis of the attenuation, but there is still a release of local cytokines and chemotactic factors that may cause the rupture of the normal function of the epithelial barrier (Sansonetti, P. J. 1991 Rev. Infect. Dis. 13: 285-292).
The viruses are also studied. The fact that the poliovirus type 1 and the attenuated strain of Sabin use the M cells to cross the epithelial barrier, make them important candidates for oral vaccines to send foreign antigens in man. Vaccinia and other poxviruses are assimilated by mucosal surfaces but their interaction with the M cells is still unknown (Report of the Expert Panel VI: Concerted efforts in the field of mucosal immunology 1996 Vaccine 14: 644-664).
Many complex carbohydrates of natural origin stimulate the cells of the immune system and the reticulum-endothelium system (Davis, S. E. 1975 Am. Chem. Soc. Sympos. Series 15, Jeanes A. Hodge J. Eds. Am. Chem. Soc. Washington D.C.). Among these are the polymers of plants and fungi as the glucans, dextrans and lentinans, all of which are glucose polymers, and the mannans, among which are found the glucomannans and the galactomannans. Also found are the levans and xylans (Tizard, I. R. et al. 1989 Mol. Biother 1:290-296). The activity of many of these polyglycans on macrophages (having glucan and mannan receptors) include the induction of phagocytosis and the secretion of cytokines, leucotriens and prostaglandines. The lentinan, a glucan that is common in mushrooms, stimulates the cell and antibody response in sheep eythrocytes while levan is mytogenic for B cells and a macrophage activator (Simon, P. M. 1994 Exp. Opin. Invest. Drugs 3 (3):223-239).
The acemannan is a mannan composed of mannose with O acetylations in approximately 8 out of every 10 hexose-rests. It is extracted as a major component of the mucilaginous substance or gel of the Aloe barbadensis Miller leaf, medicinal plant used throughout history. Different tests in vitro indicate that the manans activate the monocytes and macrophages inducing the production of interferon-xcex3, factor-xcex1 of tumoral necrosis, colony stimulator factor of monocytes and granulocytes, IL-1xcex2 and IL-6 (Peng, S. Y. et al. 1991 Mol.Biother. 3: 79-87). The acemannan potentiates the generation of cytotoxic T lymphocytes (CTL) (Womble, D. et al. 1988 Int. J. Immuno-pharmacol. 10:967-974), the cytotoxic activity of Natural Killer (NK) cells (Marshall G. D. et al. 1993 J. Immunol. (part II) 150: Abstr 1381), and also, slightly, the in vitro human alloresponse.
The increase of the cytotoxic activity and the secretion of xcex3 interferon supports the antiviral and antitumoral therapeutic use of acemannan. Its antiretroviral activity was evidenced in animals in the case of feline leukemia (Sheets, M. A. et al. 1991 Mol. Biother. 3: 41-45). Clinical assays in AIDS and cancer patients are currently in course.
Patents have recently been applied in relation to the use of the acemanan as an adjuvant for vaccines. (McAnalley, B. H. EP 0 619 117 A2, Nordgrem, R. M. WO 93/14195), but in none of the two cases the nasopharyngeal use of the acemannanis protected. In both patents the antigens are inoculated by the systemic route (subcutaneous and intramuscular). In relation to the mucosal route, the first patent shows poor results in the oral use of a acemannan formulation. The results obtained with an acemannan oral formulation (shown in the first patent) may be considered poor compared to those obtained by the systemic inoculation route. As previously explained, the adjuvants may have different effects in the different mucous sites due to the physiology of the assimilation of the antigen and the different environment in each route, that may affect the activity of the immunological potentiator (Report of the Expert Panel VI: Concerted efforts in the field of mucosal immunology 1996 Vaccine 14: 644-664). Besides the differences between the mucosal routes, different results have also been obtained when the same adjuvant is used systemically or through mucosal routes. This is the case of the most common systemic adjuvant used, the aluminum hydroxide. This adjuvant has not been more effective than the PBS when mice are inoculated through the oral and nasal routes with antigens for which it is the traditional adjuvant for systemic usexe2x80x94as for example the tetanus toxoidxe2x80x94(Alpar H. O. et al. 1992 Int. J. of Pharm. 88: 335-344). Therefore, it is not obvious that a systemic adjuvant is also necessarily a mucosal adjuvant.
None of the compositions referred in a patent application of a combined vaccine composition containing HBVs antigen contains acemannan and they haven""t been used them through the nasopharyngeal route of inoculation (Tyrrell, A. et al. WO 93/24148).
The main technical feature of a patent about the fusion of proteins to LTB to use those fusion proteins as therapeutic and preventive vaccines differs from our main technical feature cause in the description of the preferred embodiments it is stated that the fused protein comprises heat-labile enterotoxin B subunit (LTB) and a protein which is heterologous to heat-labile enterotoxin. The heterologous protein includes, for example, antigens (proteins or polypeptides). In the scope of our claims we consider that this kind of formulations do not meet the characteristics of our formulations (F. Yukio EPA 0 418 626 A2) cause the antigens to be fused in our preparations do not contain as a result of this fusion the adjuvant as a fused protein and to be used it should be mixed with the acemannan.
A lot of articles in the art discuss strategies of delivery to mucosal tissues for antigens in liposomes. These liposomes are immunogenic in mice and guinea pigs at a low dose and without any other adjuvant (Ilona Idxc3xa4np{umlaut over (aa)}n-Heikkilxc3xa4 et al. Vaccine 13 (16), 1995: 1501-1508);(Daiichi pharm co. LTD, Database WPI, AN 96-379257). Other antigen delivery systems has been used (M, Yukata et al. DE 196 27 392 A1). Our work consider the mixture of soluble proteins delivered in antigen delivery systems with acemannan as a way to increase the immune response until levels achieved throught systemic inoculations.
The present invention is related to a vaccine formulation for nasopharyngeal administration, having as its main components subunit particulated antigens and the acemannan, in adequate proportions.